Hcm Associated Cardiac Troponin I Mutations Alter Cardiac Troponin Function, Contractile Properties And Modulation By Pka Mediated Phosphorylation

We recently reported on the altered structure-function relationship for two HCM-associated cardiac troponin I (cTnI) mutations, R146G and R21C (located in the inhibitory-peptide and cardiac-specific N-terminus of cTnI, respectively), and how this resulted in the cardiac dysfunction. Both mutations significantly increased Ca2+ binding affinity to cTn (KCa) and the affinity of cTnC for cTnI (KC-I). In isolated myofibrils, both mutations increased Ca2+ sensitivity of tension (pCa50) while maximal tension (TMAX) was maintained. PKA phosphorylation of cTnI resulted in decreased pCa50 for cTnIWT exchanged myofibrils, but not for either mutation. PKA phosphorylation accelerated the early, slow-phase relaxation for cTnIWT myofibrils, especially at Ca2+-levels that the heart operates in-vivo. Importantly, this effect was blunted for cTnIR146G and cTnIR21C exchanged myofibrils, as was PKA-mediated reduction in KC-I. Molecular-dynamics simulations of cardiac troponin (cTn) suggested both mutations inhibit formation of intra-subunit contacts between the N-terminus and the inhibitory-peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation, suggesting this may be the mechanism of disrupting modulation of contractile properties for these mutants. In ongoing studies, we are examining two additional HCM-associated mutations, cTnIP83S or cTnIA158V, located in the I-T arm and switch-peptide of cTnI, respectively. Both mutations also significantly increased KCa and KC-I, and blunt the PKA effects on KC-I, similar to cTnIR146G and cTnIR21C. Preliminary kinetics/mechanics results indicated that TMAX was also maintained for cTnIP83S and cTnIA158V exchanged myofibrils, but pCa50 was significantly increased only for cTnIA158V myofibrils. Additionally, preliminary measurements suggest these mutants also blunt the ability of PKA phosphorylation to decrease pCa50 and accelerate the early, slow-phase relaxation. We are currently performing molecular-dynamics simulations of cTn to understand the structural basis of these effects for both mutations.